Native metabolomics identifies the rivulariapeptolide family of protease inhibitors

The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.


TABLE OF CONTENTS
probe to obtain the correlations of the major components of that fraction in about 14 hours.
The raw data was processed (phase correction, baseline correction, removal of t1 noise) with MestreNova Version 12.0 and the major correlations were manually peak picked.

In silico MS/MS annotation
Comparison of the SMART results revealed that eight of ten predicted structures were identical and five or respectively six out of ten compounds were AHP-cyclodepsipeptides.
In depth analysis of the top ten predicted structures with NPClassifier 4 (https://npclassifier.ucsd.edu/) showed that all, but one structure were classified as cyclic depsipeptides and all of the structures were further classified as polyketides, suggesting a NRPS/PKS hybridic pathway for the biosynthesis of the main compound in fraction 1-1 (and 2-1, respectively).
We annotated the Ahp-cyclodepsipeptide candidates as members of the subfamily rivulariapeptolides followed by their molecular weight if CANOPUS 5 detected the substructure element 'delta-lactam' or 'piperidone' with a posterior probability of > 99% in combination with the detection of substructural elements such as 'pyrrolidinecarboxamide', 'N-acylpyrrolidines', and 'proline and derivatives' with a posterior probability of > 90%, indicative of the characteristic N-butyrylated proline residue. All MS/MS of isolated compounds were deposited as public spectra within GNPS 6 along with confirmed activity of the respective compounds as protease inhibitors utilizing GNPS tags.
Relative and absolute configurations of the rivulariapeptolides S5 The absolute configurations of the amino acids were determined by UHPLC-MS analysis of the acid hydrolysates of 2 (rivulariapeptolide 1155) and its PDC oxidation product (Supplementary Figure 41a). Oxidation followed by acid hydrolysis liberated glutamic acid, allowing the assignment of the C-3 position of the Ahp (3-amino-6-hydroxy piperidone) unit. The analysis revealed L-configuration for C-3 as well as for the C-alphas of every further amino acid in the rivulariapeptolides (as it is the case for all previously reported cyanobacterial Ahp-cyclodepsipeptides). The relative stereochemistry of the Ahp moiety was determined to be (3S*, 6R*)-Ahp based on NMR spectroscopic data (Supplementary Figure 41b). In the Ahp ring system, NOESY correlations were observed between the diaxially oriented H-3 (δ 3.78 ppm), H-5a (δ 1.71 ppm), and the equatorially oriented H-6 (δ 5.07 ppm, br s (J < 1 Hz)) 7 . Thus, the hydroxyl group of C-6 had to be axially oriented and this axial orientation of the 6-OH group is responsible for the downfield shift of H-4a (δ 2.42 ppm). Furthermore, the NOESY correlations between H-3 (δ 3.78 ppm) and the equatorial H-4b (δ 1.58 ppm) supported the assigned relative stereochemistry. Together with the results from the PDC oxidation and Marfey's analysis we determined the absolute configuration to be (3S, 6R)-Ahp in line with the stereochemical assignments in other Ahp-cyclodepsipeptides such as tutuilamide A and molassamide, respectively 8,9 . The geometry of the Abu olefinic bond was determined as "Z" based on HMBC and NOE correlations in DMSO-d6. A four-bond HMBC correlation between H3-4 and C-1 of Abu indicates a "w" configuration for bonds between these atoms and, therefore, the Z-geometry for the double bond 10 . NOESY correlations from H3-4 of Abu (δ 1.51) to H-2 of Thr-1 (δ 4.59) and H3-4 of Thr-2 (δ 1.11) further support this configuration.

Pyridinium dichromate (PDC) oxidation
Rivulariapeptolide 1155 (Compound 2, 0.5 mg) was dissolved in CH2Cl2 (0.5 mL) and mixed with PDC (2.0mg). After stirring at rt for 5h, the reaction was quenched by shaking Docking studies were performed to to rationalize the bioactivity of the isolated compounds and to explore the potential for structure modifications that could yield lead structures for therapeutic interventions. The isolated compounds were docked by induced-fit, inside the S7 binding pocket of alpha-chymotrypsin (PDBID 4Q2K) and all were found to have a similar binding mode (Supplementary Figure 45b, d, e). Crystal structures of Ahpcyclodepsipeptides in complex with serine proteases by others 8,11 indicate that inhibition is based on a substrate-like binding mode in which distinct amino acid residues occupy the S-and S'-pockets, however, proteolytic cleavage does not occur, because the AHP moiety occupies an important part of the active site pocket. Furthermore, it has been demonstrated that the residue immediately following the Ahp unit should bind in the S1 specificity-determining pocket 12,13 . Structure-activity relationships towards elastase inhibition revealed that the 2-amino-2-butenoic acid (Abu) moiety incorporated within the macrocycle, contributes to potent elastase inhibition 8,14 . This is in accordance with our experimental results where the replacement of the Abu (compound 2) moiety for Leu (1) led to a fivefold decrease in potency towards elastase, but simultaneously to a more than threefold increase in potency towards chymotrypsin. The previously reported importance of a polar functionality in the side chain towards more potent elastase inhibition could not be corroborated by our data 11,14 . Masking, the secondary alcohol of the threonine in the side chain by an esterification with N-butyryl proline (2) instead of bearing the free polar hydroxyl group (4) increased the potency towards elastase. Binding to proteinase K, however, seems to be more favorable with less rigid and bulky side chain substituents as found in compounds 5 and 6.

Structural analysis
We modeled the rivulariapeptolides peptides bound to α-chymotrypsin, based on the crystal structure (PDB ID 4GVU) of the macrocyclic peptide lyngbyastatin 7, which shares a chemical scaffold with the rivulariapeptolides, bound to elastase, which has 37% sequence identity with alpha-chymotrypsin and a similar three-dimensional structure. A crystal structure of alpha-chymotrypsin (PDB ID 4Q2K) was aligned with the elastase structure, using the Molecular Operating Environment (MOE, version 2019.01) software, transferred the lyngbyastatin 7 molecule to the chymotrypsin structure, and energyminimized the resulting bimolecular complex. We then used ChemDraw integration with MOE to sketch each of the rivulariapeptidolides and energy-minimized each resulting S8 complex. Minimization was performed using the Amber10:EHT force-field, no constraints were applied, and the convergence criterion was set to RMS of 0.1 kcal mol -1 Å 2 .

Protein Expression and Purification
Cloning and protein purification of TasA Next day, cells were harvested by centrifugation (7,000 × g, 10 min, 4 °C) and resuspended in buffer A (50 mM Tris, 150mM NaCl, pH 8), and then centrifuged again.
The pellets were kept at −80 °C until purification or processed after 15 min. Cells were resuspended in buffer A, sonicated on ice (3 × 60s, 80% amplitude) and centrifuged (15,000 × g, 10 min, 4 °C). The supernatant was discarded, pellet was resuspended in buffer A supplemented with 2 % Triton X-100 and then incubated at 37 °C for 20 min.

Cloning and protein purification of Chitosanase (Csn)
Plasmid pET22b-csn was constructed by amplifying csn gene from B. subtilis NCIB3610.

Cloning and protein purification of DEV3pro
The

Cloning and protein purification of Pim1
The pLIC-SGC construct containing Pim-1 with a 6xHis tag, TEV cleavage site and an R250G mutation was kindly provided by E. Meggers, Philipps-Universität Marburg. After the mutation back to wild type, the vector was transformed into BL21 (DE3) gold cells.
After an overnight expression in SLB media at 291.15 K, cells were harvested by centrifugation and disrupted with a high-pressure homogenizer (Emulsiflex C5, Avestin).
The lysate was centrifuged and the supernatant loaded onto a nickel affinity column Afterwards, the plasmid was transformed into One Shot® TOP10 electrocompetent E.
Positive transformants were selected on LB-agar plates containing 100 µg/mL ampicillin.
Following purification of the plasmid, the correct insertion of cutA1 into pASK was verified   1-80 eV). The scatter-plot on the right shows the relative peak height (apo vs. holo) over the different CID energy. b) displays the XICs of the chymotrypsin x molassamide complex (top left) and apo chymotrypsin (bottom left) over stepwise increased HESI AUX temperatures (40-300 °C). The scatter-plot on the right shows the relative peak height (apo vs. holo) over the different temperature steps. c) displays the XICs of the chymotrypsin x molassamide complex (top left) and apo chymotrypsin (bottom left) over stepwise increased relative S-lense radiofrequency (RF) level (10-90%). The scatter-plot on the right shows the relative peak height (apo vs. holo) over the different RF levels. Source data are provided as a Source Data file.